Treatment of Medium-Chain Acyl-CoA Dehydrogenase Deficiency

ABSTRACT

The present invention provides for methods and compositions for treating medium chain acyl-CoA dehydrogenase deficiency. It is based, at least in part, on the discovery that phenylbutyrate can serve as a substrate for medium chain acyl-CoA dehydrogenase. In non-limiting embodiments, phenylbutyrate and/or another source of phenylacetate is administered as a chaperone treatment to patients suffering from medium chain acyl-CoA dehydrogenase deficiency.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of International Patent ApplicationPCT/US2013/036739, filed Apr. 16, 2013, and claims priority to U.S.Provisional Application Ser. No. 61/624,864 filed Apr. 16, 2012, to bothof which priority is claimed and the contents of which are incorporatedherein in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant NumberHD056004 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

INTRODUCTION

The present invention provides for methods and compositions in whichphenylbutyrate or other source of phenylacetate is used to treatmedium-chain acyl CoA dehydrogenase deficiency.

BACKGROUND OF THE INVENTION MCADD

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency (MCADD) is aninborn error of fatty acid metabolism and rivals phenylalaninehydroxylase deficiency (PKU, Phenylketonuria) as the most commonbiochemical genetic disorder in the United States. The overall frequencyof the disease has been estimated at 1:6,500 to 1:17,000 in Caucasiansof mostly Northern European ancestry. The MCAD gene is located onchromosome 1p31 and contains 12 exons (Zhang et al., Biochemistry31:81-9, 1992). An MCAD A985G point mutation has been identified in 90%of the alleles in the MCAD gene in deficient patients. This mutationsubstitutes a glutamate for a lysine at position 304 (K304E). Anothercommon apparently silent mutation, A11610 (V362V) is found in someindividuals in exon 11. Multiple mutations have been identified in thesecond allele 18 individuals having one A985G allele: G127A (E17K),T233C (I53T), 244 insertion T (D79X), G253T (G60C), T320C (L82S), A430T(K144X), 431-434 deletion AGTA (R123X), T464C (M130T), T499C (S142P),A589G (K172E), C631T (P186S), C683A (T203N), G799A (G242R), or G881C(R269T). All were found to be clinically affected. Heterozygotes areusually asymptomatic.

MCADD patients are asymptomatic at birth but are at risk for episodes ofacute, life threatening metabolic decompensation. These usually firstoccur between 3 and 24 months of age but can occur at any age inassociation with physiologic stress such as fasting or infection, fever,or strenuous exercise. The mortality rate during an acute crisis inpreviously undiagnosed patients can be as high as 20%. MCADD can now beidentified pre-symptomatically, nearly eliminating mortality due to thisdisease. However, there is currently no drug used to treat MCADD, andthe standard of care requires lifelong dietary monitoring. Significantmorbidity still occurs; for example, patients frequently requirehospitalization for IV glucose therapy in the face of reduced oralintake during illnesses. Thus a medication capable of relieving themetabolic block would be of great benefit to these patients.

The Acyl-CoA Dehydrogenase Gene Family

MCAD is a member of the acyl-CoA dehydrogenase (ACD) family of enzymes,a group of structurally similar enzymes that catalyze theα,β-dehydrogenation of acyl-CoA esters and transfer electrons toelectron transferring flavoprotein (ETF). Biochemical and immunologicalstudies have identified at least 9 distinct members of this enzymefamily, each with a characteristic substrate specificity (Ikeda et al.,J. Biol. Chem. 258: 1066-1076, 1983; Ikeda et al., J. Biol. Chem. 260:1311-1325, 1985; Ikeda et al., J. Biol. Chem. 258: 9477-9487, 1983; Izaiet al., J. Biol. Chem. 267:1027-1033, 1992; Rozen et al., Genomics 24:280-287, 1994; Willard et al., Arch. Biochem. Biophys. 331:127-133,1996; Nguyen et al., Mol. Genet. Metab. 77: 68-79, 2002; Zhang et al.,Biochem. Biophys. Res. Commun. 297:1033-42, 2002). Very long, ACD9,long, medium and short chain acyl-CoA dehydrogenases (VLCAD, ACD9, LCAD,MCAD, and SCAD) catalyze the first step in the β-oxidation cycle withsubstrate optima of 16, 16:1, 14, 8 and 4 carbon chains respectively.Isovaleryl-CoA dehydrogenase (IVD), short/branched chain ACD (SBCAD),and isobutyryl-CoA dehydrogenase (IBD) catalyze the third step inleucine, isoleucine and valine metabolism, respectively (Ikeda et al.,J. Biol. Chem. 258: 9477-9487, 1983; Rozen et al., Genomics 24: 280-287,1994; Nguyen et al., Mol. Genet. Metab. 77: 68-79, 2002; Ikeda et al.,J. Biol. Chem. 258: 1077-1085, 1983; Ikeda et al., Methods Enzymol 166:360-73, 1988; Ikeda et al., Methods Enzymol 166: 374-89, 1988;Finocchiaro et al., J. Biol. Chem. 262: 7982-9, 1987; Binzak et al.,Biochimica et Biophysica Acta 1382: 137-142, 1998), while glutaryl-CoAdehydrogenase (GDH) functions in the lysine catabolic pathway. Whilemature VLCAD and ACD9 are dimers, the other ACDs are homotetramers, witheach monomer containing a non-covalently bound flavin adeninedinucleotide molecule (FAD) as a prosthetic group (Ikeda et al., J.Biol. Chem. 258: 1066-1076, 1983; Ikeda et al., Biochemistry 24:7192-7199, 1985; Ikeda et al., J, Biol. Chem. 260: 1326-1337, 1985). Allof these enzymes have been purified to homogeneity from tissue orrecombinant sources, and antisera have been produced (Ikeda et al., J.Biol. Chem. 260: 1311-1325, 1985; Ikeda et al., J. Biol. Chem. 258:9477-9487, 1983; Izai et al., J. Biol. Chem. 267:1027-1033, 1992; Nguyenet al., Mol. Genet. Metab. 77: 68-79, 2002; Ikeda et al., J. Biol. Chem.258: 1077-1085, 1983; Binzak et al., Biochimica et Biophysica Acta 1382:137-142, 1998, Finocchiaro et al., J. Biol. Chem. 263: 15773-80, 1988;Aoyama et al., Journal of Clinical Investigation 95: 2465-2473, 1995;Mohsen et al., Gene 160: 263-267, 1995). All of the ACDs have beenpostulated to share a common ordered BiBi type kinetic mechanism (Ghislaet al., Biochemistry 23: 3154-3161, 1984; Thorpe et al., FASEB 9:718-725, 1995; Thorpe et al., C, Schaller R A, Mohsen A-W and Vockley J:The acyl-CoA dehydrogenases: Some mechanistic aspects. University ofCalgary Press, Calgary, Canada, 1997, pp. 597-604). Reduction of theenzyme via dehydrogenation of the substrate occurs when a glutamateresidue acting as a catalytic base abstracts the a-carbon proR hydrogenof substrate as a proton. The β-carbon proR hydrogen transfers as ahydride to N-5 of FAD and a stable intermediate, the charge transfercomplex (CTC), is formed. Following establishment of the CTC,reoxidation of the enzyme occurs when ETF interacts with the enzyme torelease the product.

In the proposed enzyme mechanism of all ACD reactions, the formation ofa charge-transfer complex is postulated to be initiated with theabstraction of the substrate pro-R α-hydrogen as a proton by an activesite glutamate base. The pro-R β-hydrogen of the acyl moiety of thesubstrate is then transferred as a hydride to the N-5 of the flavin ring(Ikeda et al., J. Biol. Chem. 260: 1311-1325, 1985; Ikeda et al.,Biochemistry 24: 7192-7199, 1985). In the absence of an electronacceptor, ETF, the transfer of the substrate pro-R β-hydrogen as ahydride to the N-5 of the FAD is postulated to be incomplete, creating aresonant donor/acceptor hybrid species, or the “charge-transfercomplex”, with the acyl-CoA substrate/product remaining tightly bound tothe enzyme (Ikeda et al., J. Biol. Chem. 260: 1311-1325, 1985; Ikeda etal., Biochemistry 24: 7192-7199, 1985). The abstraction of the pro-Rβ-proton and a transfer of the pro-R β-hydride to form thecharge-transfer complex has also been postulated to occur in a concertedfashion (Thorpe et al., FASEB 9: 718-725, 1995; Thorpe et al., C:Electron-transferring flavoproteins. In: Chemistry and Biochemistry ofFlavoenzymes. CRC Press, Inc., Boca Raton, Fla., 1991, pp 471-486;Thorpe et al., Biochemistry 18: 331-337, 1979). This interaction betweenthe substrate, the active site base, and the oxidized FAD disrupts theextended π-electron system of the FAD isoalloxazine ring, quenching itscharacteristic absorbency at 445-450 nm and causing a new absorptionband to appear at 580 nm. Under physiological conditions, thecharge-transfer complex interacts with ETF, and the reduced ETF andenoyl-CoA are released as the end products to regenerate the oxidizedenzyme (Ikeda et al., Biochemistry 24: 7192-7199, 1985; Ikeda et al., J.Biol. Chem. 260: 1326-1337, 1985; Thorpe et al., FASEB 9: 718-725, 1995;Thorpe et al., C, Schaller R A, Mohsen A-W and Vockley J: The acyl-CoAdehydrogenases: Some mechanistic aspects. University of Calgary Press,Calgary, Canada, 1997, pp. 597-604; Massey V: A simple method for thedetermination of redox potentials. In: Yagi K (ed) Flavins andFlavoproteins 1990, Walter deGruyte, New York, 1991, pp 59-86).Important clues for deciphering the mechanism of interaction between ETFand its redox partners have been obtained with the crystallization ofhuman MCAD:ETF (PDB: 2A1T) and M. methylotrophus trimethylaminedehydrogenase:ETF (PDB: 1UDY) ternary complexes (Leys et al., Nat StructBiol 10:219-25, 2003; Toogood et al., J. Biol. Chem. 279: 32904-12,2004; Toogood et al., J. Biol. Chem. 280: 30361-30366, 2005). Thecrystallization of ETF with these two different redox partners suggeststhat ETF could bind to its partner without the former necessarily beingreduced or being in the binary complex form with substrate. They alsoshow that the identity of the recognition/anchor site of ETF for itsbinding partners is the same for the different enzymes. Anotherimportant feature that these two complexes share is that the β-subunitcontaining the recognition-anchor domain acts to provide a staticbinding force, while domain II, which is responsible for electrontransfer, is so highly dynamic that it is absent from the electrondensity data collected.

Sodium Phenylbutyrate

Sodium phenylbutyrate (“Buphenyl®”) is a commercially available drugused for treating PKU, where phenylbutyrate removes excess ammonia byconjugating its active form, phenylacetate, with glutamine. Buphenyl® isconverted into this active form after one cycle of 13-oxidation.

SUMMARY OF THE INVENTION

The present invention provides for methods and compositions for treatingMCADD. It is based, at least in part, on the discovery thatphenylbutyrate can serve as a substrate for MCAD. In non-limitingembodiments, phenylbutyrate or another source of phenylacetate isadministered as a chaperone treatment to patients suffering from MCADD.The present application also provides for compositions comprisingphenylbutyrate or another source of phenylacetate, for use in thepreparation of a medicament for treating MCADD.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic showing a proposed overall pathway for metabolism ofphenylbutyrate to its final metabolite.

FIG. 2: Monitoring the formation of the charge transfer complex withpurified MCAD upon addition of increasing amounts of octanoyl-CoA. Theabsorbance maxima at ˜370 nm and ˜447 nm are reduced and a new peakcentered at ˜570 nm appears with addition of increasing substrate.Selected scans are shown with octanoyl-CoA concentration at 0, 3.25,7.1, 10.8, 15.6, 18.0, 21.5, and 28.2 μM. The inset shows the kineticsof these changes. Enzyme concentration was 25.2 μM. Equation for thedecrease at 447 nm is:y=−1×10⁻⁹x⁶+1×10⁻⁷x⁵−2×10⁻⁶x⁴+2×10⁻⁵x³−0.0003x²−0.008x+0.3489. Equationfor the increase at 570 nm is:y=6×10⁻¹⁰x⁶−5×10⁻⁸x⁵+1×10⁻⁶x⁴−2×10⁻⁵x³+0.0003x²+0.0003x+0.0008.

FIG. 3: Monitoring the formation of the charge transfer complex withpurified MCAD upon addition of increasing amounts of phenylbutyryl-CoA.The absorbance maxima at ˜370 nm and ˜447 nm are reduced and a new peakcentered at ˜570 nm appears with addition of increasing substrate.Selected scans are shown with phenylbutyryl-CoA concentration at 0, 4.2,8.3, 16.3, 24.1, 31.6, 40.7, and 80.2 μM. The inset shows the kineticsof these changes. Enzyme concentration was 25.2 μM. Equation for thedecrease at 447 nm is: y=5×10⁻¹⁰x⁵−1×10⁻⁷x⁴+9×10⁻⁶x³−0.0002 x²−0.0061x+0.3707. Equation for the increase at 570 nm is:y=−1×10⁻¹⁰x⁵+3×10⁻⁸x⁴−3×10⁻⁶x³+7×10⁻⁵x²+0.0012 x+0.0036.

FIG. 4: Detailed proposed pathway of metabolism of phenylbutyrate to itsactive form, phenylacetate.

FIG. 5: Stick representation of MCAD active site residues and ligandswith phenylbutyryl-CoA modeled in place of octanoyl-CoA. The crystalstructure of pig MCAD with bound octanoyl-CoA (PDB: 3MDE, (23)) was usedto create the model using MOE modeling software. The E376 carboxylate isthe active site catalytic base responsible for the substrate C2 protonabstraction to initiate catalysis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for treatingsubjects with MCADD with phenylbutyrate and/or another source ofphenylacetate.

A subject may be a human or non-human subject such as a dog, cat, horse,pig, cow, sheep, goat, rodent, rabbit, etc..

In certain non-limiting embodiments, phenylbutyrate is administered as asalt, for example, but not limited to, sodium phenylbutyrate. In aspecific, non-limiting embodiment, Buphenyl® is used.

In certain non-limiting embodiments, the source of phenylacetate is acomposition comprising phenylacetate. For example, in one specificnon-limiting embodiment, the composition is a mixture of sodium benzoateand sodium phenylacetate (e.g. Ammonul®), which may be administeredintravenously (for example, to subjects for which oral administrationwould be problematic).

In another specific non-limiting embodiment, a prodrug of phenylbutyrateand/or of phenylacetate, for example triphenylbutyrylglycerol (alsoknown as glycerol phenylbutyrate, benzenebutanoic acid,1′,1″-(1,2,3-propanetriyl) ester and RAVICTI™), AN-113 (also known asbutyroyloxymethyl-4-phenylbutyrate), or combinations thereof, is used.

Phenylbutyrate, for example as a salt, may be administered by a routeselected from the group consisting of oral, intravenous, intrathecal,intraperitoneal, nasal, pulmonary, rectal, vaginal, subcutaneous,intradermal, or intramuscular. It may be administered via a reservoir.

In certain, non-limiting embodiments, the present invention provides fora method of treating a subject with MCADD comprising administering, tothe subject, an effective amount of phenylbutyrate, for example as aphenylbutyrate salt, as a regular therapy regimen. In a regular therapyregimen, phenylbutyrate is administered on a regular basis, for example,but not limited to: each time the subject eats, with each meal, once aday, twice a day, three times a day, four times a day, five times a day,six times a day, every 24 hours, every 12 hours, every eight hours,every six hours, or every four hours. The regular therapy regimen may beadministered for a duration that may be, for example, but not limitedto: continuous (without a planned termination), one year, one month, twomonths, three months, four months, five months, six months, sevenmonths, eight months, nine months, ten months, eleven months, one week,two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks,eight weeks, nine weeks, ten weeks, eleven weeks, twelve weeks, thirteenweeks, fourteen weeks, fifteen weeks, sixteen weeks; twenty weeks;twenty eight weeks, one day, two days, three days, four days, five days,six days, seven days, eight days, nine days, ten days, eleven days,twelve days, thirteen days, fourteen days, fifteen days, sixteen days,seventeen days, eighteen days, nineteen days, twenty days, twenty-onedays, twenty-two days, twenty-three days, twenty-four days, twenty-fivedays, twenty-six days, twenty-seven days, twenty-eight days, twenty-ninedays, thirty days, or thirty-one days.

In certain, non-limiting embodiments, the present invention provides fora method of treating a subject with MCADD comprising administering, tothe subject, an effective amount of phenylbutyrate, for example as aphenylbutyrate salt, when the subject is at metabolic risk. The subjectis at metabolic risk as a result of one or more of the following:infection; a planned or passed interval without food (“a fastinginterval”) of at least six hours, or at least about 8 hours, or at leastabout 10 hours, or at least about 12 hours, or at least about 18 hours,or at least about 24 hours; and/or an intercurrent illness leading toincreased metabolic energy demand and/or reduction of food intake. Thephenylbutyrate, for example as a phenylbutyrate salt, may beadministered, for example, but not by limitation: each time the subjecteats, with each meal, once a day, twice a day, three times a day, fourtimes a day, five times a day, six times a day, every 24 hours, every 12hours, every eight hours, every six hours, every four hours, every twohours, or every hour. In specific non-limiting embodiments, thetreatment interval may be: for the duration of the period over which thesubject is at metabolic risk; for one day, two days, three days, fourdays, five days, six days, seven days, eight days, nine days, ten days,eleven days, twelve days, thirteen days, fourteen days, fifteen days,sixteen days, seventeen days, eighteen days, nineteen days, twenty days,twenty-one days, twenty-two days, twenty-three days, twenty-four days,twenty-five days, twenty-six days, twenty-seven days, twenty-eight days,twenty-nine days, thirty days, or thirty-one days.

In certain non-limiting embodiments, the phenylbutyrate may beadministered as sodium phenylbutyrate (e.g., Buphenyl®). In specificnon-limiting embodiments, sodium phenylbutyrate may be administeredorally. The maximum daily dose of sodium phenylbutyrate is 20 grams perday. In specific non-limiting embodiments, sodium phenylbutyrate may beadministered at a dosage of 450-600 mg/kg/day in subjects weighing lessthan 20 kg. In specific non-limiting embodiments, sodium phenylbutyratemay be administered at a dosage of 9.9-13.0 g/m²/day for subjectsweighing 20 kg or more. In a specific, non-limiting embodiment, thedaily dose of sodium phenylbutyrate may be divided into 4, 5, or 6dosages. In specific non-limiting embodiments, sodium phenylbutyrate maybe administered at a dose of less than 450 mg/kg/day. In specificnon-limiting embodiments, sodium phenylbutyrate may be administered at adose of between 200-400 mg/kg/day. In specific non-limiting embodiments,sodium phenylbutyrate may be administered at a dose of 4-8 g/m²/day. Inspecific non-limiting embodiments, sodium phenylbutyrate may beadministered at a dose of 4-6 g/m²/day. In specific non-limitingembodiments, sodium phenylbutyrate may be administered at a dosage of0.5g. In specific non-limiting embodiments, sodium phenylbutyrate may beadministered at a dosage of 1 g. In specific non-limiting embodiments,sodium phenylbutyrate may be administered at a dosage of 2 g.

In certain non-limiting embodiments, a prodrug of phenylbutyrate and/orof phenylacetate, for example, triphenylbutyrylglycerol (i.e.,RAVICTI™), AN-113, or combinations thereof, is administered according tothe present application. In specific non-limiting embodiments, theprodrug may be administered orally. In certain non-limiting embodiments,the maximum daily dose of RAVICTI™ is 17.5 mL, or 19 grams per day. Inspecific non-limiting embodiments, RAVICTI™ may be administered at adosage of 4.5-11.2 mL/m²/day. In specific non-limiting embodiments,RAVICTI™ may be administered at a dosage of about 5-12.4 g/m²/day. In aspecific, non-limiting embodiment, the daily dose of RAVICTI™ may bedivided into 2, 3, 4, 5, or 6 dosages. In certain non-limitingembodiments, the RAVICTI™ is administered to a patient who is naïve tophenylbutyrate. In certain non-limiting embodiments, RAVICTI™ isadministered to a patient who has previously been treated with sodiumphenylbutyrate, wherein the prodrug is administered in an amount equalto 0.86 times the total daily dosage of phenylbutyrate previouslyadministered to treat the patient.

In specific non-limiting embodiments, RAVICTI™ may be administered at adose of less than 19 g/day. In specific non-limiting embodiments,RAVICTI™ may be administered at a dose of between 0.5-4.0 mL/m²/day. Inspecific non-limiting embodiments, RAVICTI™ may be administered at adose of between 0.1-0.5 mL/m²/day. In specific non-limiting embodiments,RAVICTI™ may be administered at a dose of 0.5-4 g/m²/day. In specificnon-limiting embodiments, RAVICTI™ may be administered at a dose of0.1-0.5 g/m²/day. In specific non-limiting embodiments, RAVICTI™ may beadministered at a dosage of 0.5 g/m²/day. In specific non-limitingembodiments, RAVICTI™ may be administered at a dosage of 1 g/m²/day. Inspecific non-limiting embodiments, RAVICTI™ may be administered at adosage of 2 g/m²/day. In specific non-limiting embodiments, RAVICTI™ maybe administered at a dosage of 5 g/m²/day. In specific non-limitingembodiments, RAVICTI™ may be administered at a dosage of 10 g/m²/day. Inspecific non-limiting embodiments, RAVICTI™ may be administered at adosage of 0.5 mL/m²/day. In specific non-limiting embodiments, RAVICTI™may be administered at a dosage of 0.6 mL/m²/day. In specificnon-limiting embodiments, RAVICTI™ may be administered at a dosage of 1mL/m²/day. In specific non-limiting embodiments, RAVICTI™ may beadministered at a dosage of 2 mL/m²/day. In specific non-limitingembodiments, RAVICTI™ may be administered at a dosage of 4 mL/m²/day. Inspecific non-limiting embodiments, RAVICTI™ may be administered at adosage of 4.5 mL/m²/day. In specific non-limiting embodiments, RAVICTI™may be administered at a dosage of 5 mL/m²/day. In specific non-limitingembodiments, RAVICTI™ may be administered at a dosage of 10 mL/m²/day.

In certain non-limiting embodiments, a prodrug is administered at adosage of about 10 to about 50 fold lower than an effective dosage ofphenylbutyrate. In certain non-limiting embodiments, a prodrug isadministered at a dosage of about 15 to about 25 fold lower than aneffective dosage of phenylbutyrate. In certain non-limiting embodiments,a prodrug is administered at a dosage of about 20 fold lower than aneffective dosage of phenylbutyrate.

In certain non-limiting embodiments, a mixture of sodium phenylacetateand sodium benzoate may be administered to the subject, for example, butnot limited to, Ammonul®. In a specific, non-limiting embodiment, forsubjects weighing less than 20 kg, 0.25 g/kg of each of sodium benzoateand sodium phenylacetate may be administered daily, or, if the subjectweighs more than 20 kg, 5.5 g/m² of each of sodium benzoate and sodiumphenylacetate may be administered daily.

EXAMPLE Medium Chain Acyl-CoA Dehydrogenase Plays a Key Role inMetabolism of Phenylbutyrate Materials and Methods

Purification of Recombinant Human MCAD. Expression and purification ofrecombinant human MCAD was perforated as previously described forisovaleryl-CoA dehydrogenase with minor modifications (18). E. coliJM105 cells (Amersham Biosciences Corp; Piscataway, N.J.) containing thehuman MCAD high expression vector pKeMCAD (19) and a GroEL/GroESexpression plasmid were grown overnight in a 200-ml LB broth pre-culturethat was used to inoculate 4×2-L cultures in 2-YT broth. The cells wereleft to grow overnight at 37° C. with shaking and MCAD expression wasinduced the next morning using IPTG at a final concentration of 0.5 mMfor 3 hrs. Cells were harvested by centrifugation and resuspended at 4°C. in 2:1 weight to volume of 100 mM potassium phosphate pH 8.0, 150 mMEDTA. Cells were then lysed by sonication on ice. Including high amountsof EDTA in the cell lysis buffer can protect residues with groups, e.g.,cysteine thiols and methionine sulfide groups, vulnerable tomodification by oxygen reactive species generated during sonication cellsuspension. This was effective in improving enzyme preparationsresulting higher specific activity and consistent kinetic behavior.Cellular debris was removed by centrifugation first at 250,000×g for 60minutes each. The final supernatant was dialyzed for 4 hours withvigorous stirring in 50 mM potassium phosphate pH 8.0, at 4° C. Thesample was then loaded on a 16×40 mm DEAE Sepharose FF columnpre-equilibrated in 50 mM potassium phosphate pH 8.0, using an ÄKTAUPC-900 pump FPLC system (Amersham Biosciences Corp; Piscataway, N.J.).After washing with 300 ml of 50 mM potassium phosphate pH 8.0, MCAD waseluted with a 300 ml linear gradient from 50 to 500 mM potassiumphosphate pH 8.0. Green fractions with a 270/447 rim ratio <12containing MCAD were pooled, concentrated, and dialyzed against 25 mMpotassium phosphate, pH 8.0. Pooled fractions of essentially pure MCAD(270/447 nm ratio=5.5), were concentrated and stored at −80° C. Otherrecombinant human ACADs were similarly purified except that the protocolwas terminated after the DEAE-Sepharose column for human recombinantLCAD as the enzyme was unstable. LCAD protein purity was at about 70% atthis stage.

ETF Purification. Porcine ETF was purified as previously published (20),except that the dialysis buffer used after both the 40-60% ammoniumsulfate fractionation and DE-52 cellulose anion-exchange chromatographysteps consisted of unbuffered 15 mM dibasic potassium phosphate and 5%glycerol.

Fibroblast Cell Culture and Extract Preparation. Wild type and MCADdeficient cells (homozygous for the K304E mutation) with the designationGM085401 and GM07844, respectively, were obtained form Coriell Institutefor Medical Research, Camden, N.J. Cells were cultured in DMEM mediumsupplemented with glutamine and ampicillin and streptomycin, and 20%fetal bovine serum. Cells were harvested from a T175 flask by sonicationwith a buffer consisting of 50 mM Tris buffer and 10 mM EDTA, pH 8.0.The cell debris was removed by centrifugation and the cell free extractwas assayed for protein and enzyme activity as described below.

ETF Fluorescence Reduction Assay. The ETF reduction assay was performedusing a Jasco FP-6300 spectrofluorometer (Easton, Md.) with a cuvetteholder heated with circulating water at 32° C. The assay was otherwiseperformed as described (22), at the indicated substrate concentrations.The enzyme was diluted 1200-fold into a buffer containing 50 mM Tris, pH8.0, 5 mM EDTA and 50% glycerol, and 10 μl were used for each assay. TheETF concentration in the reaction mixture was 2 μM. Spectra Manager 2software (Jasco Inc) was used to collect data and calculate reactionrate and Microsoft Excel was used to calculate the kinetic parameters.

Phenylbutyryl-CoA Synthesis. CoASH, octanoyl-CoA, C12-CoA andphenylbutyric acid were obtained from Sigma (St. Louis, Mo.)2,6-dimethylheptanoic acid was obtained from Matreya LLC (Pleasant Gap,Pa.). The phenylbutyryl-CoA and 2,6-dimethylheptanoyl-CoA esters wereprepared by the mixed anhydride method as described previously (24) andwas purified by HPLC using a Luna 5 μm C 18(2) column (25 cm×0.46 cm)and a linear gradient (10-60%) of acetonitrile into 50 mM ammoniumphosphate, pH 5.5, at a flow rate of 1.5 mL/min over 30 min.

Monitoring the Interaction of MCAD with Substrates, Formation of thecharge transfer ternary complex was monitored by observing the increasein absorbance at the 570 nm area, concomitant with the decrease ofabsorbance at 447 nm area, of the purified MCAD in 120 mM potassiumphosphate spectrum under anaerobic conditions using a Jasco V-650Spectrophotometer. A quartz cuvette with a round top containing 0.5 mlof the purified MCAD in 120 mM potassium phosphate, pH 8.0, was sealedwith a rubber plug, and using a needle, ten alternating cycles of vacuumand argon were applied to remove oxygen. One μL at a time ofphenylbutyryl-CoA solution dissolved in water to 0.53 mM was then addedto the sample in the sealed cuvette using a 50 μl Hamilton syringeattached to an automatic dispenser. Ten seconds of equilibration timewere allowed after mixing and the sample was scanned for UV/Visiblelight absorbance at 250 to 800 nm. Final substrate concentrations variedfrom 0 to 28.2 μM for octanoyl-CoA and 0 to 80.2 μM forphenylbutyryl-CoA. All data were adjusted for the dilution resultingfrom substrate addition. Substrates were titrated, but with differentfinal concentrations as indicated in the figure legends. The “apparent”productive-binding constant (Kd app) was calculated with the followingequation:

$\frac{d}{p} = {{K_{D\mspace{11mu} {app}}\frac{1}{e - p}} + n}$

where d is the total ligand concentration, e is the total molarconcentration of enzyme, p is the fraction of enzyme sites that bindligand multiplied by e, and n is the number of binding sites. Theabsorbance at 447 nm when all enzyme sites are occupied with ligand wasdetermined separately by adding large excess of octanoyl-CoA and used tocalculate the fraction of enzyme with bound ligand at various readingsand assuming that at large excess of added substrate would equal to e.

Molecular Modeling. Computer modeling of MCAD was performed using aSilicon Graphics Fuel workstation (Mountain View, Calif.) with theInsight II 2005 software package and MOE software, from ChemicalComputing Group, Montreal, Canada, and the atomic coordinates of pigMCAD (3MDE) in the dimer form as a molecule (23). Carbon atoms atpositions C5-C8 of the octanoyl-CoA ligand, which co-crystallized withMCAD, were replaced with a phenyl group. The phenylbutyryl-CoA ligandconformation in the active site was refined using the Discover module.Human LCAD 3D structure was modeled using MCAD atomic coordinates astemplate and the Insight II modeling software.

RESULTS

Interaction of MCAD with Substrates, the Reductive Half-Reaction.Formation of the charge transfer complex, the reductive half-reaction,is evident from the spectral scans of MCAD at various phenylbutyryl-CoAconcentrations (FIGS. 2 and 3). The progressive decrease and increase ofabsorbance at 447 nm and 570 nm, respectively, are similar to thoseinduced by octanoyl-CoA. The octanoyl-CoA binding curve is sigmoidal incontrast to the phenylbutyryl-CoA binding curve, possibly reflectingdifferences in enzyme mechanism of interaction. The plot of d/p versus 1/e-p (the Stockell plot) was nonlinear. A line drawn at the straightarea of the curvewhere the substrate:subunit ratio was 1:1 estimates theapparent dissociation constant (K_(D) app) being 0.12 μM and 2.16 μM foroctanoyl-CoA and phenylbutyryl-CoA, respectively. Other mathematicalderivatives of the absorbance data all indicated that the binding sitesare non-equivalent.

Interaction of MCAD:Substrate Ternary Complex with ETF, the OxidativeHalf-Reaction. Transfer of electrons is evident from decrease in ETFfluorescence when ETF is used as the electron acceptor in the presenceof various concentrations of phenylbutyryl-CoA. The catalytic efficiencyand K_(m) for the phenylbutyryl-CoA were 0.2 mM⁻¹sec⁻¹ and 5.3 μMcompared to 4.0 mM⁻¹sec⁻¹ and 2.8 μM for octanoyl-CoA, respectively.

Molecular modeling of human LCAD shows possible accommodation of theacyl moiety of the phenylbutyryl-CoA, with the exception of residueL264, which would have one of its side chain methyl hydrogens ˜1.3 Åaway from a phenyl ring hydrogen and so would hinder binding. To test ifthe LCAD active site has enough plasticity to accommodate this potentialsubstrate, we measured its activity with LCAD using the ETF fluorescencereduction assay. While the partially purified recombinant human LCAD wasactive in the presence of various substrates including C₁₂-CoA and2,6-dimethylheptanoyl-CoA, it was not active in the presence ofphenylbutyryl-CoA. Purified SCAD, MCAD, and ACAD9 also showed noactivity with phenylbutyryl-CoA as substrate.

The ETF Fluorescence Reduction Assay of Cell Extract. ETF fluorescencereduction was observed using extracts from wild type fibroblast cells inthe presence of 30 μM of phenylbutyryl-CoA, octanoyl-CoA, orpalmitoyl-CoA. (The latter substrate was used as internal control and isa substrate of VLCAD.) Enzyme specific activity measured using thesesubstrates was 3.41 (±0.53), 4.01 (±1.34), 9.10 (±2.13) nmoles ETFreduced×min⁻¹×mg⁻¹, respectively. No activity was observed using similaramounts of extract from fibroblast cells deficient in MCAD with eitherphenylbutyryl-CoA or octanoyl-CoA. The measured enzyme specific activityof palmitoyl-CoA oxidation in extract from these cells was 3.91 (±1.34)nmoles ETF reduced×min⁻¹×mg⁻¹.

Discussion

Following the conversion of phenylbutyrate to the CoA ester, one cycleof β-oxidation is expected to result in phenylacetyl-CoA and acetyl-CoAas the final products. (FIG. 4). Phenylacetyl-CoA is hydrolyzed tophenylacetate, which becomes conjugated with glutamine and is excretedin urine (FIG. 1). An analysis of this first step in the β-oxidation ofphenylbutyryl-CoA is important because the first step in the β-oxidationof fatty acids is postulated to be rate-limiting, and thus themetabolism of phenylbutyrate to its active form, phenylacetate, may alsobe modulated by similar factors that affects energy metabolism at thesame step (25).

The effect of phenylbutyryl-CoA on the MCAD absorbance spectrum atrelatively low concentrations is monitored via the decrease ofabsorbance at 447 nm and increase of absorbance at 570 nm. This confirmsproductive binding of phenylbutyryl-CoA to MCAD in the reductivehalf-reaction with lack of product release. This effect is similar tothat induced by octanoyl-CoA binding to MCAD, and indicative of thetransfer of a proton and a hydride to the flavin ring and formation ofthe charge transfer complex, which is comprised of the enzyme, reducedFAD, and enoyl product and detected by the intense absorbance bandcentered at 570 nm.

The inset in FIG. 2 shows, however, a sigmoidal shaped curve induced byaddition of octanoyl-CoA, with an exponential change phase centering atsubunit:substrate ratio of 4:1. This corresponds to one octanoyl-CoAmolecule binding the first subunit. Such a behavior was not detectedwhen phenylbutyryl-CoA was used as substrate. This may imply positivecooperativity between the first and second subunits when octanoyl-CoA,but not phenylbutyryl-CoA, is used as substrate. Although otherinterpretations of sigmoidal behavior in this setting are possible,including presence of various MCAD forms or other effector molecules,the argument is weakened by the fact that the only difference betweenthe two reactions is the substrate itself Impurities in the substratepreparation are also not likely to induce such an effect as suchimpurities would be present at ineffectively low concentrations at thelow substrate concentrations range, between 0.25-1 and 4:1substrate:MCAD tetramer ratio. Why this kinetic behavior has not beenreported earlier is perhaps due to the method of isolation andpurification of the MCAD protein. Our current standard protocol forrecombinant protein purification includes adding high concentrations ofEDTA in the cell lysis buffer to protect residues with groups, e.g.,cysteine thiols and methionine sulfide groups, vulnerable tomodification by oxygen reactive species generated when sonication isused to break the E. coli cell wall. Using EDTA to protect residues fromoxygen radical species has proven to be effective in providing ourenzyme preparations with higher specific activity and consistent kineticbehavior (26).

Reduction of ETF by the charge transfer complex in the oxidativehalf-reaction shows that electrons from the bound phenylbutyryl-CoA canbe productively transferred to ETF and the product, phenylbutenoyl-CoA,is released to complete the reaction. In contrast, none of the otherACADs are capable of catalyzing this reaction.

Modeling of a phenylbutyryl moiety in the active site in place of theoctanoyl moiety observed in the MCAD crystal structure shows the phenylmoiety accommodated in the acyl moiety binding site pocket with aconformation perpendicular to the aromatic ring of Y375, FIG. 5. Otherresidues involved in binding the phenyl moiety include E99, A100,Leu103, and V259. Furthermore, modeling predicts that the phenyl ringpara and/or meta positions are candidate expansion sites for adding afunctional group that may improve binding, while addition at the orthoposition would prevent the derivative from binding to MCAD.

Based on the kinetic parameters of MCAD with phenylbutyryl-CoA assubstrate, individuals with MCAD deficiency are likely to experience afunctionally relevant decrease in the ability to metabolize themedication (e.g., phenylbutyrate), though indications for use in thesepatients are likely to be rare. Of note, since octanoyl-CoA has beenreported to provide thermal stability to the MCAD K304E mutant [27], itis possible that phenylbutyryl-CoA would behave similarly and may be ofbenefit in vivo in patients carrying at least one copy of this mutation.It is unknown if carriers for MCAD deficiency, a much more commonsituation, will display altered metabolism of phenylbutyrate. In otherindications where the functional effects of phenylbutyrate are less wellcharacterized, modulation of MCAD activity might be of benefit to alterdrug metabolism and/or its half-life and increase its efficacy,depending on the mechanism of action of the medication in each disorder.

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Various publications are cited herein, the contents of which are herebyincorporated by reference in their entireties.

What is claimed is:
 1. A method of treating a subject with medium chainacyl-CoA dehydrogenase deficiency comprising administering, to thesubject, an effective amount of phenylbutyrate and/or another source ofphenylacetate as a regular therapy regimen.
 2. The method of claim 1,where phenylbutyrate is administered as a phenylbutyrate salt.
 3. Themethod of claim 2, where the salt is sodium phenylbutyrate.
 4. Themethod of claim I, where triphenylbutyrylglycerol is administered to thesubject as a source of phenylacetate.
 5. The method of claim 1, where amixture of sodium benzoate and sodium phenylacetate is administered tothe subject as a source of phenylacetate.
 6. A method of treating asubject with medium chain acyl-CoA dehydrogenase deficiency comprisingadministering, to the subject, an effective amount of phenylbutyrateand/or another source of phenylacetate when the subject is at metabolicrisk.
 7. The method of claim 6, where the subject is at metabolic riskbecause of an infection.
 8. The method of claim 6, where the subject isat metabolic risk because of a fasting interval.
 9. The method of claim6 where phenylbutyrate is administered as a phenylbutyrate salt.
 10. Themethod of claim 9, where the salt is sodium phenylbutyrate.
 11. Themethod of claim 6, where triphenylbutyrylglycerol is administered to thesubject as a source of phenylacetate.
 12. The method of claim 6, where amixture of sodium benzoate and sodium phenylacetate is administered tothe subject as a source of phenylacetate.
 13. Use of a compositioncomprising phenylbutyrate and/or another source of phenylacetate in thepreparation of a medicament for treating a subject diagnosed or at riskof developing medium chain acyl-CoA dehydrogenase deficiency.
 14. Theuse of claim 13, where phenylbutyrate is a phenylbutyrate salt.
 15. Theuse of claim 14, where the salt is sodium phenylbutyrate.
 16. The use ofclaim 13, where triphenylbutyrylglycerol is the source of phenylacetate.17. The use of claim 13, where the source of phenylacetate comprises amixture of sodium benzoate and sodium phenylacetate.